PROJECT SUMMARY/ABSTRACT: Rising antibiotic resistance in bacterial pathogens highlights the urgent need to understand the molecular mechanisms by which bacteria cause infections, in order to develop effective precision-based antibiotic-sparing therapies. Gram-negative bacterial pathogens encode over 100 extracellular fibers termed chaperone/usher pathway (CUP) pili able to recognize and colonize different host tissues and habitats, a prerequisite to infection. Each CUP pilus is encoded as part of a gene cluster containing a designated periplasmic chaperone and outer membrane (OM) usher protein that facilitates assembly of tens of hundreds of structural subunits into each final pilus structure. In addition, most CUP pili are tipped by two-domain adhesins comprising: i) an N-terminal domain that recognizes a receptor with stereochemical specificity; and ii) a C-terminal pilin domain. Chaperone- subunit/adhesin complexes are formed through a donor strand complementation (DSC) interaction in which the chaperone donates steric information to promote the folding of the pilin domains and are then delivered to the OM usher which catalyzes subunit-subunit interactions via donor strand exchange (DSE). DSE occurs when an amino-terminal extension (Nte) present on each subunit completes the Ig fold of its neighbor. Pilus biogenesis catalyzed by the OM usher is a remarkably complex process involving the multiple domains of the usher functioning as a nanomachine to assemble pili tipped with an adhesin. With the support from this grant, considerable progress has been made towards elucidating the mechanism of pilus biogenesis, however, this proposal seeks to fill key knowledge gaps: understanding the molecular workings of the usher. To do this, we will elucidate three-dimensional structures of usher intermediates representing critical points in the assembly cascade: i) pilus initiation; and ii) subunit incorporation/(DSE) using X-ray crystallography and single-particle cryo-electron microscopy (Aim 1). We will elucidate the mechanisms by which both two-domain adhesins and specialized single-domain pilins activate ushers in three distinct pilus systems (Aim 2). Understanding the molecular biology of CUP pili has already led to a FimH-based vaccine that was developed to prevent recurrent uropathogenic E. coli (UPEC) urinary tract infections (UTI). This was developed based on understanding that type 1 pili tipped with FimH mediate bladder colonization. The vaccine has completed a Phase 1A/1B study and received FDA allowance for compassionate use based on promising results. In addition, rationally designed inhibitors of FimH function, termed mannosides, have been shown to be highly efficacious in treating and preventing UTI in mouse models, while simultaneously being able to selectively deplete UPEC from the mouse gastrointestinal tract reservoir. Here, small molecules that block usher function will be developed (Aim 3), which would potentially block assembly of multiple CUP pili and work synergistically with other therapeutics. Thus, structural and functional insights will be gained and integrated to develop antibiotic-sparing therapeutics that prevent UPEC colonization by blocking usher and adhesin function.